Exosomes from B cells and Dendritic cells: mechanisms of formation, secretion and targeting

Abstract

Many cell types, including dendritic cells (DC) and B cells, secrete small vesicles called exosomes. Exosomes from immune cells are thought to have immuno-regulatory functions but their precise role remains unresolved. The aim of the studies presented in this thesis was to get more insight into the factors that determine exosome formation, composition and secretion as well as to learn more about their physiological relevance. Exosomes are equivalent to Luminal Vesicles (LV) of Multi Vesicular Bodies (MVB), which are secreted when MVB fuse with the plasma membrane. Studying exosomes may therefore also help to unravel mechanisms that are important for MVB biogenesis. Incorporation of membrane proteins into the LV of MVB requires, at least for some model proteins, ubiquitination of their cytoplasmic domain. These ubiquitin tags are recognized by a sorting machinery. We demonstrate that exosomes from B cells and DC contain poly-ubiquitinated proteins, which are not integrated into the membrane and relatively enriched as compared to total cell lysates, suggesting that a subset of poly-ubiquitinated cytoplasmic proteins is incorporated into the MVB. Furthermore, we present a detailed analysis of the proteome of B cell exosomes using mass spectrometry. In total 681 different proteins were identified which may be important for protein sorting at MVB or exosome targeting or funtion. Furthermore we found that in exosomes, major histocompatibility complex class II (MHC class II) is associated with large protein networks, which include at least 24 other proteins. These protein networks may reflect a role for protein interactions in exosome biogenesis at MVB. Alternatively, on exosomes, protein clusters may enhance the function of proteins that are important for binding to and signalling in target cells. In addition we show that DC secrete exosomes into the immune synapse upon engagement with cognate CD4+ T cells and that MHC class II is transferred from the DC to T cells via exosomes. Exosomes are directionally targeted to T cells both by secretion in the immune synapse and through specific binding to activated but not to resting T cells, indicating that only those T cells that are activated as a result of cognate DC/ T cell interaction may acquire DC exosomes. These results resolve a long standing question of how MHC II and other membrane constituents are transferred from antigen presenting cells to T cells. Finally we directly studied the molecular mechanism for MHC class II targeting to exosomes and demonstrated that this process requires ubiquitination of its b-chain. In addition, we show that, regardless of the stimulus, DC maturation is accompanied by a decrease in ubiquitination of total cellular MHC class II, consistent with the idea that MHC class II surface expression is stabilized by shutting down ubiquitin-mediated intracellular retention. We demonstrate that during cognate DC/T cell interaction DC increase the sorting of MHC class II to exosomes and at the same time elevate cell surface MHC class II, indicating that ubiquitin mediated trafficking of MHC class II is highly regulated and that exosomes may derive from a specialized subset of MVB.